Approximately 40,000 cases of oral squamous cell carcinoma (OSCC) are diagnosed each year in the United States. Surgery, chemotherapy, and radiation are the mainstays of treatment for advanced OSCC, but frequently result in disfigurement and adverse cytotoxicities. Recurrent disease, leading to death, is associated with development of chemotherapy and/or radiation resistance. Efforts to combat OSCC have been severely hindered by an incomplete understanding of the mechanisms of disease progression and a lack of molecular markers that can be used to predict responsiveness to chemotherapy and radiation. Our long-term goal is to determine the role that gene mutations play in OSCC, to apply this knowledge towards risk stratification in patients, and to develop therapeutic strategies to overcome the negative consequences of these mutations. We and others have reported the mutational landscape of OSCC, identifying the procaspase-8 gene as one of the most commonly mutated genes in this disease. Mutations in the coding region for procaspase-8 zymogen were identified in 8 percent of patient tumors, a finding confirmed by analysis of 302 OSCC tumors by The Cancer Genome Atlas (TCGA). Wild-type caspase-8 is known to mediate death receptor-mediated apoptosis. In functional studies of four OSCC-associated, procaspase-8 mutants, we determined that the mutant proteins potently inhibit death receptor-mediated apoptosis, and a representative mutant interfered with formation of the death inducing signaling complex (DISC). The procaspase-8 mutants also conferred enhanced resistance to cisplatin and radiation. Inhibition of the DNA damage response proteins ATR and ATM reversed cisplatin and radiation resistance, respectively, in cells with mutant procaspase-8. We hypothesize that OSCC- associated procaspase-8 mutations promote resistance to death ligands, chemotherapy, and radiation, while defining a substantial subpopulation of patients who may benefit from treatment with ATR or ATM inhibitors. To test this hypothesis, we propose three Specific Aims. Aim 1 will utilize innovative OSCC models to investigate the impact of a broad panel of OSCC-associated procaspase-8 mutant proteins on DISC formation, apoptosis, and necroptosis following death receptor activation. Aim 2 will determine whether the mutant proteins confer resistance to cisplatin or radiation in vitro and in vivo, and will compare clinical outcomes of OSCC patients harboring wild-type versus mutant procaspase-8 using specimens from an ongoing Phase II clinical trial incorporating concurrent cisplatin/radiation treatment. In Aim 3, i vitro and in vivo studies will determine the potential of ATR/ATM inhibition as a strategy for reversing chemotherapy and radiation resistance. Results from our studies will determine the significance and consequences of frequent procaspase-8 mutation on OSCC responsiveness to death receptor agonists, chemotherapy, and radiation, and the mechanisms responsible for these effects. We also expect that our findings will lay the foundation for biomarker-driven clinical trials evaluating the value of ATR or ATM inhibition in patients with procaspase-8 mutations.